Previous pilot studies had identified markedly biased usage of codon pairs within ORFs of E. coli,S. cerevisiae and C. albicans, suggestive that codon pair usage may generally regulate translation elongation in some way. In this work, comprehensive analyses of coding sequences of multiple genomes revealed that biased usage of codon pairs is universal, and that a tetranucleotide sequence spanning the last position of the P-site codon and all nucleotides in the A-site codon underlies this selection. Furthermore, in S. cerevisiae, S. pombe, B. subtilis and members of the gamma-protobacteria family (including E. coli), interactions between the A-site tRNA and the 3rd position nucleotide of the P-site codon, act as a key selective force driving codon pair selection. Analysis revealed A-site tRNA sequences in the anticodon stem loop and the amino acid acceptor stem that positively and negatively contribute to codon pair selection in a variety of prokaryotes. tRNA availability, coupled with usage of codons in ORFs, is yet another key regulation point in elongation. Previous studies in prokaryotes have suggested that the encounter of repeated runs of rare codons by elongating ribosomes can lead to processivity errors (e.g. ribosomal drop off, frameshifting) due to the depletion of charged tRNA levels. However, the effects of such ‘polycodon’ sequences upon eukaryotic elongating ribosomes are unknown. In this work, reporter mRNAs containing both rare and common polycodon sequences were expressed and the consequential effects on gene expression observed. Several key findings emerged from this work. Firstly, repeated runs of rare and common codons inserted in frame between two fused reporter genes caused reduced expression of the 3’ ORF reporter activity. Evidence was obtained indicating a novel proteolysis event acted between the two reporter enzyme domains, as well as preliminary evidence for ribosomal drop off. Finally, a novel +1 frameshifting motif involving CGG polycodon sequences was identified.